Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation - PubMed (original) (raw)

Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation

Yen K Lieu et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Hematopoietic stem cells (HSCs) have a unique capacity to undergo self-renewal and multi-lineage differentiation to provide a lifetime supply of mature blood cells. By using conditional knockout technology, we disrupted the c-myb proto-oncogene specifically in adult bone marrow (BM) to demonstrate that this transcription factor is a regulator of self-renewal and multi-lineage differentiation of adult HSCs. Targeted disruption of the c-myb gene resulted in a critical depletion of the HSC pool. In addition, BM hematopoiesis in adult mice was impaired, resulting in profound reductions of various hematopoietic lineages including neutrophilic, monocytic, B lymphoid, erythroid, and, unexpectedly, megakaryocytic cells. Serial BM transplantation into lethally irradiated recipient mice indicated an essential role for c-myb in the self-renewal process. Furthermore, in vitro functional assays demonstrated that deletion of the c-myb gene leads to a slightly reduced proliferative capacity and an aberrant and accelerated differentiation of HSCs. In addition to long-term HSCs, functional studies also show that c-myb plays a critical role in short-term HSCs and multi-potential progenitors. Collectively, our data indicate a critical role for c-myb in adult BM hematopoiesis and in self-renewal and multi-lineage differentiation of adult HSCs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

c-myb is required for adult BM hematopoiesis and maintenance of HSCs. (A) Cellularity of BM and spleen of pIpC-induced _myb_f/f/MxCre mutant (KO) and litter-mate control _myb_f/f (CON) mice. (B) PCR analysis of genomic DNA from BM and spleen of KO and CON mice. (C) Absolute number of cells in various BM lineages is depicted on a bar graph. (D) Representative 2-color flow cytometric or histogram analysis shows the percentages of various BM populations. (E) Expression of c-myb in hematopoietic stem cells as determined by semiquantitative RT-PCR. (F) Absolute number of cells in the hematopoietic progenitor (Lin−c-Kit+Sca-1−, LKS−) and stem cell (LKS+) compartments as well as LT-HSC, ST-HSC, and MPP cells. (G) Representative 2-color flow cytometry showing the percentages of LKS−, LKS+, LT-HSC, ST-HSC, and MPP cells. (*, P < 0.001; †, P < 0.05. Numbers are presented as mean ± SEM; n = 25 mice.)

Fig. 2.

Fig. 2.

Hematopoietic stem cells have an intrinsic requirement for c-myb to maintain their populations. Percentages of experimental CD45.2 (blue bar) and competitive CD45.1 (yellow bar) lineage (A) and stem cells (D) in the BM of induced _myb_f/f/MxCre chimeras (KO) and controls. Representative histogram and 2-color flow cytometric analysis showing the percentages of CD45.2 and CD45.1 cells in various BM lineages (B) and stem cells (E) of the first transplanted mice after pIpC administration. (C) DNA analysis of total BM cells from mixed chimeric mice after pIpC administration. For controls and conditional KO for all lineages except whole blood-derived erythroid cells, n = 10 and n = 11, respectively. For whole blood erythroid cells, n = 3 for both groups. Data are expressed as mean ± SEM. Error bars are shown for CD45.2 cells only. (*, P < 0.001; †, P < 0.05.)

Fig. 3.

Fig. 3.

c-myb is required for self-renewal of hematopoietic stem cells. (A) BM DNA from pIpC-treated first transplants (_myb_f/+/MxCre or _myb_f/f/MxCre chimeras) that were pooled to use as donor cells to reconstitute lethally irradiated mice to generate second transplants. (B) Percentages of CD45.2 and competitor CD45.1 cells in total BM of second transplanted recipient mice. (C) PCR analysis of genomic DNA from BM of second transplants. (D) Representative 2-color flow cytometric analysis showing the percentages of CD45.2 and CD45.1 cells in LKS−, LKS+, LT-HSC, ST-HSC, and MPP cells of second transplants. (n = 4 in each group.)

Fig. 4.

Fig. 4.

Disruption of c-myb in hematopoietic stem cells leads to impaired proliferation and accelerated differentiation. Following 48 h IFN treatment to induce disruption of c-myb, the purified LT-HSCs, ST-HSCs, and MPPs were labeled with BrdU (C) and then stained for surface expression of lineage markers and c-Kit (D). Shown are the representative 2-color flow cytometric analysis of CD11b (y-axis) and CD41 surface antigens for LT-HSCs (A) (n = 3), ST-HSCs (B, Top) (n = 3), and MPPs (B, Bottom) (n = 4). Representative 2-color flow cytometric analysis of BrdU incorporation (C) and c-Kit expression (D) for LT-HSCs (n = 6) are shown. (E) Cells in the lin-c-Kit+Sca-1+ (LKS+) compartment of pIpC-induced KO mice were analyzed for surface expression of CD11b, CD41, Gr-1, and c-Kit. n indicates the number of experiments. Data are expressed as mean ± SEM. (*, P < 0.05; †, P < 0.01.) IFN (KO+) or pIpC (KO)-treated _myb_f/f/MxCre.

Fig. 5.

Fig. 5.

Disruption of the c-myb gene results in impaired HSC growth and multi-lineage differentiation on methylcellulose and altered gene expression. (A) Hematopoietic colony assays performed using purified LT-HSCs, MPPs, and LKS+ cells. The number of colonies from the 3 groups was normalized to the untreated control, which was set to 100%. Data are expressed as mean ± SD, n = 3 experiments. (B) PCR analysis of genomic DNA from 18 h IFN-treated LKS+ cells before plating on methylcellulose containing dishes (Top) and cells from colonies growing on dishes after 12 d (Bottom). Semiquantitative RT-PCR analysis of gene expression of LT-HSCs (C) and ST-HSCs (D) after 18 h of IFN treatment. Results reflect ≥3 sorted experiments. GEMM, granulocytic, erythroid (E), monocytic, and megakaryocytic (Meg) colonies; GM, granulocytic and monocytic colonies. (*, P < 0.001.)

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